Morphological or isozyme markers related to physiological maturation and deteriorative processes are important in the evaluation of seed quality. Two experiments were conducted to examine the possibility of using isozymes as indicators of quality in tobacco seed lots and fruit appearance as an indicator of physiological maturity in tobacco cultivars, based on the physiological and biochemical changes of the seeds. Cultivars CSC 444 and CSC 221 of tobacco fruits were harvested at various maturity stages and their physiological quality was assessed by germination, first count, germination speed index, time to reach 50% germination, cumulative average germination, and seedling emergence. We also assessed the activity of catalase (EC 1.11.6.1 – CAT), esterase (EC 3.1.1.1 – EST), isocitrate dehydrogenase (EC 1.1.1.41 – IDH), malate dehydrogenase (EC 1.1.1.37 – MDH), alcohol dehydrogenase (EC 1.1.1.1 – ADH), endo-β-mannanase (EC 3.2.1.78), and heat-resistant proteins during the process of maturation. Six lots of cultivar CSC 444 were used to differentiate the quality levels between the lots, and their characterization was determined by germination and vigor tests. In addition, we evaluated the enzymatic activity of CAT, EST, ADH, MDH, and heat-resistant proteins. During maturation of the fruits from the partially dark stage, we observed a progressive increase in germination and seed vigor. We concluded that appearance of the fruit is an indicator of fruit maturity and quality in tobacco seeds. The enzymatic profile of ADH matches the physiological potential of the seeds, based on germination and emergence tests. Thus the ADH enzyme indicates the optimum stage to harvest fruits. In the EST and CAT enzymatic pattern analysis, we observed higher activity of these enzymes in lots with lower physiological quality. So the CAT and EST enzymes are biochemical indicators that can assess the deterioration of tobacco seed lots.